PROJECT SUMMARY Genotyping of cancer has become the standard of care, where specific mutations in cancer-driving genes have been shown to indicate sensitivity or resistance to targeted treatments. Sampling of cell-free DNA (cfDNA) in blood plasma could provide a non-invasive means of performing this genotyping, but current methods are too complex, slow (3-14 days), and expensive (hundreds of dollars) to make use of this information in a manner that would have a major clinical impa ct. To widely impact cancer care, a minimally invasive monitoring assay must be inexpensive enough (~$30) that it can be ordered repeatedly, and must be fast enough (<3 hrs) that it can seamlessly integrate into the current pace of clinical decision making. This Phase I STTR proposal seeks to evaluate the feasibility of DropWorks? continuous emulsion digital PCR (cdPCR) technology for rapid, low-cost, non-invasive genotyping of cancer through measurement of cell-free DNA in blood plasma. The high level of automation in this technology greatly reduces the complexity of the workflow, allowing it to be placed in typical hospital laboratories and obviating the requirement for specialty laboratory staff. Its simplified, continuous nature has the potential to greatly reduce turnaround time (~30 minutes) and cost (~$30). This tool could provide clinicians with low-cost, high frequency information on genotype status and trends for a wide range of mutations known to be indicative of therapeutic response, improving outcomes through clearer selection and adaptation of treatment. The project objective will be accomplished in partnership with Dana-Farber Cancer Institute, which has developed a set of validated cfDNA genotyping assays for non-small cell lung cancer using digital droplet PCR. In this project, assays for ex19 deletion, L858R, and T790M of EGFR will be the focus, as these have specific near-term relevance to treatment. The project will determine whether the existing cdPCR technology can be automated (reducing clinical expertise requirements and cost) and adapted to DFCI?s specific cancer assays (Aim1), and whether the specificity and sensitivity of the assays meet those for the validated digital PCR approach in constructed samples (Aim 2) and a small human pilot trial (Aim 3). A Phase 2 proposal would extend this work by transforming the instrument into a robust clinical tool, confirming sensitivity and specificity for NSCLC in a wider human trial, designing a full clinical trial, and expanding the assay set to other mutations and cancers.